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Am J Physiol Heart Circ Physiol 291: H3065-H3075, 2006. First published July 21, 2006; doi:10.1152/ajpheart.00353.2006
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Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo

Yingbo Yang,1 Wei-Zhong Zhu,2 Mei-ling Joiner,3 Rong Zhang,1 Carmine V. Oddis,1 Yue Hou,1 Jinying Yang,3 Edward E. Price,4 Linda Gleaves,1 Mesut Eren,1 Gemin Ni,3 Douglas E. Vaughan,1,5 Rui-Ping Xiao,2 and Mark E. Anderson3,6

1Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee; 2Laboratory of Cardiovascular Science, National Institutes of Aging, National Institutes of Health, Baltimore, Maryland; 3Department of Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa; 4Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center; 5Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee; and 6Department of Physiology, Carver College of Medicine, University of Iowa, Iowa City, Iowa

Submitted 2 April 2006 ; accepted in final form 17 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca2+ stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca2+ content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca2+ content by CaMKII. PLN–/– mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN–/– mice, indicating the involvement of a Ca2+-dependent pathway. AC3-I and AC3-C mice were bred into a PLN–/– background. Loss of PLN increased and equalized SR Ca2+ content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend (P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca2+ content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.

myocardial infarction; isoproterenol; programmed cell death; phospholamban

CALCIUM IS A UBIQUITOUS SIGNAL for regulating cellular functions, including survival and death (4). The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is dynamically activated by cellular Ca2+ (10) and serves as a downstream signal transduction molecule for many conditions marked by Ca2+ mobilization, including myocardial infarction (MI) and activation of the beta-adrenergic receptor (beta-AR) pathway by isoproterenol (Iso) (40). Left ventricular (LV) dilation and dysfunction are two elements that define an adverse remodeling process that is associated with cardiomyocyte death and increased mortality in patients with structural heart disease (18). CaMKII inhibition reduces adverse LV remodeling after Iso and MI in part by improving the mechanical properties of surviving ventricular myocytes (40), but it is unknown whether CaMKII inhibition can also contribute salutary effects in clinically relevant disease models by reducing cell death.

Recently, CaMKII was identified to be a facilitator of cardiomyocyte apoptosis in response to Iso in vitro (43). The antiapoptotic actions of CaMKII inhibition in vitro were mirrored by sarcoplasmic reticulum (SR) Ca2+ depletion using thapsigargin, a potent antagonist of the SR Ca2+-ATPase (SERCA2a) (43). These findings suggest that CaMKII might also be important for regulating apoptosis in vivo under conditions of increased beta-AR stimulation. However, the relationship of in vitro-to-in vivo findings for CaMKII signaling in apoptosis is far from certain, because of the vastly different conditions of intracellular Ca2+ in cultured cells compared with the beating heart. We used a mouse model with cardiomyocyte-delimited transgenic expression of a CaMKII inhibitory peptide (AC3-I, I is for inhibitor) to test the potential role of CaMKII in apoptosis in vivo. A second line of transgenic mice expressed an inactive, scrambled version of AC3-I (AC3-C, C is for control). AC3-C mice and wild-type (WT) littermates were used as controls. Myocardial function is equivalent in AC3-I, AC3-C, and WT hearts and isolated ventricular myocytes at baseline and after Iso (40). Apoptosis was not evident above control levels in AC3-I mice after Iso, whereas AC3-C and WT mice exhibited significant increases above control levels in response to Iso injection, indicating that the previously established proapoptotic role of CaMKII in isolated cells was also evident in vivo.

MI is the most common cause of adverse LV remodeling, heart failure, and excessive mortality in patients with structural heart disease (18). MI and heart failure are marked by neurohumoral activation (3), upregulation of CaMKII (17), and increased apoptosis (25). Previous cellular studies showing that CaMKII inhibition reduced catecholamine-triggered apoptosis (43) suggested that CaMKII inhibition might also be effective in reducing apoptosis during MI. We took advantage of our in vivo model to study the effects of CaMKII inhibition on apoptosis after MI. We studied AC3-I, AC3-C, and WT mice after surgical MI to test whether CaMKII was a significant regulator of apoptosis during infarction. Apoptosis was significantly reduced in AC3-I compared with AC3-C and WT mice.

Phospholamban (PLN) is a negative regulator of cytoplasmic Ca2+ uptake into the SR and is a phosphorylation target for CaMKII (5). PLN phosphorylation by CaMKII and SR Ca2+ content are significantly reduced in AC3-I transgenic mouse ventricular myocytes (40), whereas cardiomyocyte SR Ca2+ content is increased independently of CaMKII inhibition in PLN–/– mice (35). Taken together, these findings suggest the hypothesis that PLN is an important molecular determinant for the antiapoptotic actions of CaMKII inhibition. On the other hand, PLN–/– mice are a model for enhancing SR Ca2+ and myocardial function, and PLN ablation can improve adverse LV remodeling in some mouse models of cardiomyopathy (23, 29), suggesting that PLN–/– mice do not have enhanced apoptosis at baseline. We found that PLN–/– mice did not have increased apoptosis at baseline but showed greatly enhanced susceptibility to apoptosis induced by Iso, compared with WT type littermate controls. Iso-stimulated apoptosis in PLN–/– hearts was due to increased Ca2+ because it was prevented by verapamil pretreatment. These results support a strong association between PLN, myocyte Ca2+, and apoptosis in vivo while suggesting that therapeutic strategies for increasing SR Ca2+ uptake may also increase vulnerability to apoptosis.

To test whether CaMKII inhibition was antiapoptotic in the absence of PLN, we interbred AC3-I and AC3-C transgenic mice with PLN–/– mice and subjected these mice to MI and Iso stimulation. Conventional pharmacological tools for controlling SR Ca2+, such as ryanodine and thapsigargin, are incompatible with in vivo studies. One advantage of our genetic strategy is that PLN ablation eliminates differences in SR Ca2+ content between AC3-I- and AC3-C-expressing hearts by increasing SR Ca2+ content to an equivalent level (35). CaMKII inhibition failed to reduce apoptosis after MI in the absence of PLN, whereas a protective effect of CaMKII inhibition persisted after Iso, albeit with a trend (P = 0.07) toward increased Iso-dependent apoptosis in AC3-I-expressing mice with PLN ablation compared with AC3-I-expressing mice with normal PLN expression. These findings show that CaMKII is a downstream signal for apoptosis during beta-AR stimulation and MI and suggest that PLN is an important molecular target for the antiapoptotic actions of CaMKII inhibition in the beating heart.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mice. Transgenic mice with CaMKII inhibition (AC3-I) and transgenic control mice (AC3-C) were generated by myocardial-targeted overexpression of mini-genes encoding AC3-I or AC3-C, as described (40). For some experiments, we bred AC3-I and AC3-C mice into a PLN–/– background (19) and used the fifth to seventh generations of male and female PLN–/–, AC3-I x PLN–/– and AC3-C x PLN–/– mice (8–12 wk of age) for this study. All experiments were approved by Vanderbilt University and University of Iowa Animal Care Committees.

Iso treatment. A single high dose of Iso (150 mg/kg ip) or an identical volume of saline as a vehicle control was injected into mice. Verapamil (50 mg/kg ip) was given 15 min before Iso for some studies. Transverse LV sections from three to eight hearts in each experimental group were used for histological assays, as previously described (40).

MI. MI surgery was performed on mechanically ventilated (tidal volume 0.5 ml, 120 breaths/min), anesthetized mice (33 µg/g ip pentobarbital), as previously described (40). Sections 1 mm below the ligation suture line were used for apoptosis measurements. Sham MI surgery omitted the coronary artery ligation but was otherwise similar to the MI surgery. After surgery, the animals were extubated from mechanical ventilation and allowed to recover for 2–5 h before they were killed.

Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Mice were killed by cervical dislocation. We weighed the mouse and rapidly exposed the heart. While the heart was beating in situ, we injected 5,000 U of heparin and 1 ml of 2 M KCl into the right ventricle to prevent coagulation and to arrest the heart in diastole. We then perfused the heart with 20 ml of PBS and 20 ml of 10% formalin by injection through the right ventricle to wash out blood cells. The heart was then fixed in 10% formalin for ~24 h before being further processed, embedded, and sectioned into serial 7-µm-thick tissue sections. The serial tissue sections from the LV equator region were used for apoptosis measurement by using terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay. We performed TUNEL assays using In Situ Cell Death Detection Kit (Roche), according to the manufacturer’s instruction. Tissue sections were permeated with proteinase K (20 ug/ml in 10 mM Tris·Hcl, pH 7.4) for 20 min (37°C) and stained with labeling solution (including rhodamine-labeled 2-deoxynucleotide 5'-triphosphate and DNA polymerase). We pretreated the tissue sections with DNase I (100 U/ml) for 15 min for positive controls, whereas DNA polymerase was omitted for negative controls. 4,6-Diamidino-2-phenylindole (1 ug/ml, Vector) was used for nuclear staining, and myocytes were identified by expression of enhanced green fluorescent protein (eGFP) (40). We confirmed that the measured TUNEL-positive cells were cardiomyocytes by only counting TUNEL-stained nuclei encompassed by eGFP. Tissue sections were examined with an epifluorescent microscope (Olympus BX41). In Iso- or saline-treated hearts, a mid-LV cross section was divided into quarters, and the number of apoptotic myocytes in each quarter was counted under x400 magnification. After MI, the apoptosis rate was much higher, so nine random fields (x200 magnification) within the ischemic areas were examined, and the myocyte apoptosis rate was reported as the average number of apoptotic myocytes per high power field. Only cells with positive TUNEL-stained nuclei that were clearly identified as myocytes were counted and expressed as the fraction of the approximate number of cells per field. Investigators were blinded to the treatment and genetic identity of the mice in all measurements.

DNA laddering. DNA laddering was performed on hearts after MI surgery, where the burden of apoptosis was large compared with hearts from Iso-treated mice. Hearts were minced, washed three times with PBS, and frozen in liquid nitrogen. Aliquots were thawed in 720 µl of lysis buffer (10 mM Tris·HCL, pH 8.0, 100 mM NaCl, 25 mM EDTA, and 0.5% SDS) and quickly homogenized on ice using a glass-Teflon homogenizer. The tissue was digested with proteinase K at a final concentration of 1.0 mg/ml at 37°C overnight and then incubated with DNA-free RNase at 37°C for an additional hour. Sodium chloride was added to a final concentration of 1.3 M from a 5 M stock, and precipitate was removed by high-speed centrifugation. The supernatant was then extracted with 1.0 ml of 3 M NaCl-Tris-EDTA (TE; pH 8.0)-equilibrated phenol-chloroform-isoamyl alcohol (25:24:1), reextracted with chloroform-isoamyl alcohol alone, and then precipitated overnight in 2 vol of ethanol. The resulting DNA was washed in 70% ethanol and dissolved in TE (pH 8.0). After quantification with a spectrophotometer, 5 µg of DNA were loaded into 1.8% agarose gel containing 0.5 µg/ml ethidium bromide. DNA electrophoresis was carried out in Tris-Borate-EDTA buffer at 80 V for 1–2 h. DNA ladder formation was visualized under ultraviolet light (8). Investigators were blinded to the treatment and genetic identity of the mice in all measurements.

Echocardiography. Echocardiography was performed using a 15-MHz high-frequency transducer (Sonos 5500 Agilent) in unanesthetized mice as previously described (28).

Statistics. The null hypothesis was evaluated with Student’s t-test or ANOVA with {alpha} = 0.05, as appropriate. All values are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CaMKII inhibition reduces Iso-stimulated cardiomyocyte apoptosis. Catecholamines can increase cellular Ca2+ and CaMKII activity (40), leading to myocyte apoptosis in vitro (43). Catecholamines are increased in vivo during ischemia (31) and heart failure and critically drive adverse clinical outcomes under these conditions (3). We treated AC3-I, AC3-C, and WT mice with Iso or saline injection to test whether CaMKII inhibition protects against catecholamine-induced myocyte apoptosis in vivo. Our previous studies showed that the beta-AR density and the downstream protein kinase A activity, measured as Ser16 PLN phosphorylation, were equivalent in AC3-I, AC3-C, and WT mouse hearts (40). Thus differences in Iso response between AC3-I and control mice are due to reduced CaMKII activity.

Hearts from AC3-I mice treated with a single dose of Iso were significantly (P = 0.017) resistant to apoptosis compared with WT and AC3-C controls (Fig. 1), as assayed by TUNEL staining. Apoptosis was not different between saline-treated AC3-I, AC3-C, or WT mice (Fig. 1). The overall rate of apoptosis was low with this protocol, perhaps accounting for the lack of significant difference in TUNEL-positive staining between saline and Iso treatment for AC3-I, AC3-C, or WT mice. These findings indicate that CaMKII inhibition can significantly protect against, or even prevent, Iso-induced myocyte apoptosis in vivo, consistent with in vitro data (43).


Figure 1
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  		Fig. 1. Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition protects against apoptosis by isoproterenol (Iso) in vivo. A: photomicrographs showing terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining in AC3-I and AC3-C left ventricular (LV) transverse sections 2 h after Iso injection. Calibration bar indicates 15 µm. DAPI, 4,6-diamidino-2-phenylindole. B: summary data showing prevention of Iso-mediated apoptosis (5–8 hearts/group) in AC3-I mice (*P = 0.017) compared with AC3-C and wild-type (WT) mice. There were no differences in TUNEL staining in any group after saline injection (3–5 hearts/group).

 
CaMKII inhibition reduces cardiomyocyte apoptosis in ischemia. Intracellular Ca2+ overload contributes to myocyte death in ischemia, and, early in MI, apoptosis is a major mode of myocyte death (12). CaMKII is a downstream signal for cellular calcium change (7); however, the potential effect of CaMKII inhibition on apoptosis in ischemia is unknown. To test the potential role of CaMKII in ischemia-induced myocyte apoptosis in vivo, we surgically ligated the left coronary artery in AC3-I and AC3-C mice. Myocyte apoptosis was significantly lower (P = 0.018) in AC3-I hearts compared with AC3-C hearts 2 h after MI (Fig. 2, A and B), whereas sham surgery failed to elicit TUNEL-positive staining in AC3-I (n = 5) or AC3-C (n = 5) mice (data not shown). We performed DNA laddering assays, which complemented and confirmed the results of TUNEL staining by showing reduced apoptosis in AC3-I hearts after MI surgery (Fig. 2C).


Figure 2
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  		Fig. 2. CaMKII inhibition protects against apoptosis 2 h after myocardial infarction (MI) surgery. A: photomicrographs from AC3-C and AC3-I LV transverse sections are positioned side by side, labeled, and aligned vertically for comparison. Calibration bar indicates 15 µm. B: summary data (3–5 hearts/group) quantifying TUNEL-positive cardiomyocytes 2 h after MI surgery. AC3-I hearts showed a significant reduction in TUNEL-positive staining compared with AC3-C hearts (*P = 0.018). C: DNA laddering is apparent in AC3-C but not in AC3-I hearts 2 h after MI.

 
Surgical MI ultimately causes cell death of all cardiomyocytes dependent on the ligated artery, as evidenced by our previous report that AC3-I, AC3-C, and WT mice had similar areas of scar formation 3 wk after MI surgery (40). To test whether CaMKII inhibition could protect against apoptosis later than 2 h after coronary artery ligation, we repeated TUNEL staining measurements 5 h after MI surgery. CaMKII inhibition continued to significantly (P = 0.002) protect myocytes from apoptosis 5 h after MI compared with AC3-C or WT controls (Fig. 3). These findings show that CaMKII inhibition in vivo significantly inhibits or delays ischemia-induced myocyte apoptosis for at least 5 h, a time window with potentially important relevance for patients with acute coronary artery occlusion (15).


Figure 3
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  		Fig. 3. CaMKII inhibition protects against apoptosis 5 h after MI surgery. A: photomicrographs from AC3-C and AC3-I LV transverse sections, arranged as in Fig. 2, 5 h after MI surgery. B: summary data are from 3–5 hearts/group and show a significant reduction in TUNEL-positive staining in AC3-I hearts compared with AC3-C and WT hearts ({dagger}P = 0.002).

 
Our previous work showed that the size of the surgical infarction is large and consistently reproducible (40). To validate these previous results and test whether the apparent beneficial effects of CaMKII inhibition could be an artifact of inconsistency of the MI surgery, we used the percent area of TUNEL staining in LV transverse sections as an index of the ischemic burden in each of the treatment groups. Although the density of TUNEL staining was less in AC3-I compared with AC3-C and WT hearts (Figs. 2 and 3), the cross-sectional area with identifiable TUNEL staining was not different between AC3-I, AC3-C, and WT hearts (Fig. 4), suggesting that the ischemic burden was similar in all treatment groups.


Figure 4
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  		Fig. 4. Equivalent ischemic areas in AC3-I, AC3-C, and WT hearts after MI surgery, as assessed by TUNEL staining. LV cross sections show TUNEL staining in AC3-I (A) and AC3-C (B) hearts after MI surgery. Yellow lines demarcate TUNEL-positive ischemic area from nonischemic tissue. Calibration bar indicates 1 mm. C: area of TUNEL-positive staining was measured in 3–5 hearts/group and expressed as a percentage of total area 5 h after MI surgery. There were no differences in percent area with TUNEL-positive staining between any of the groups.

 
PLN ablation increases beta-AR-stimulated apoptosis by a Ca2+-dependent mechanism. PLN is a phosphorylation target for CaMKII, and the AC3-I mice have significantly reduced CaMKII-dependent PLN phosphorylation and reduced SR Ca2+ content (40). On the other hand, PLN–/– mice have elevated SR Ca2+ load (35) and significantly increased apoptosis after Iso (P < 0.001), compared with WT littermates (Fig. 5). Excess apoptosis in PLN–/– mice was due to intracellular Ca2+ overload because it was prevented by pretreatment with verapamil (Fig. 5). PLN–/– mice did not have more apoptosis than WT littermates under control (i.e., saline treatment) conditions (Fig. 5). These findings show that PLN ablation significantly increased myocardial susceptibility to apoptosis during beta-AR stimulation without affecting basal apoptosis rates, and they raise the question of whether PLN is important for the antiapoptotic actions of CaMKII inhibition in response to Iso.


Figure 5
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  		Fig. 5. Increased apoptosis in phospholamban knockout (PLN–/–) mice. A: photomicrographs showing TUNEL staining 2 h after Iso in WT littermates, PLN–/– mice, and PLN–/– mice pretreated with verapamil. Calibration bar indicates 15 µm. B: summary data show TUNEL-positive cells from 3–5 hearts/group. {dagger}P < 0.001 compared with WT and verapamil-pretreated mice; *P = 0.01 compared with WT saline-treated mice.

 
PLN–/– mice with CaMKII inhibition. To test whether CaMKII inhibition can protect against the enhanced susceptibility for Ca2+-dependent myocardial apoptosis seen in PLN–/– mice, we interbred AC3-I and AC3-C mice with PLN–/– mice (Fig. 6). The SR Ca2+ content of ventricular myocytes from these interbred mice was previously shown to be increased to an equal level in AC3-I- and AC3-C-expressing cells (35), suggesting that residual effects of CaMKII inhibition in AC3-I-expressing mice are independent of reduced SR Ca2+ content. The AC3-I x PLN–/– mice had slower heart rates compared with AC3-C x PLN–/– mice, consistent with the known effects of CaMKII inhibition to reduce cardiac pacemaker activity in vitro (33) and in vivo (40). The LV fractional shortening was modestly but significantly less in AC3-I x PLN–/– compared with AC3-C x PLN–/– mice (Fig. 6, A–C), possibly due to slower heart rates in AC3-I-expressing mice. Neither the AC3-I x PLN–/– nor the AC3-C x PLN–/– mice showed evidence of cellular or cardiac hypertrophy (Fig. 6, D and E), cardiac fibrosis, or increased TUNEL staining (not shown) at baseline compared with WT littermates, indicating that interbred PLN–/– mice do not have overt myocardial disease. This is consistent with our finding that PLN–/– mice do not have increased apoptosis at baseline, compared with WT controls (Fig. 5).


Figure 6
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  		Fig. 6. Baseline characteristics in AC3-I x PLN–/– and AC3-C x PLN–/– mice. A: representative M-mode echocardiographic tracings and summary data for heart rate (B) and LV fractional shortening (C). Summary data are from 10–12 mice/group; {dagger}P = 0.005 and *P = 0.01. D: representative micrographs of coronal and transverse cardiac sections. Calibration bar indicates 1 mm. Summary data from 8 mice/group show no significant differences (P = 0.6) in heart weight/body weight. E: representative micrographs of LV myocardium. Calibration bar indicates 10 µm. Summary data from 4 hearts/group using 200–300 myocytes/group show no significant difference in cell size (P = 0.3).

 
Apoptosis in PLN–/– mice with CaMKII inhibition. CaMKII inhibition significantly protected against apoptosis after Iso even in the absence of PLN because AC3-I x PLN–/– mice had significantly less apoptosis (P < 0.001) than AC3-C x PLN–/– mice (Fig. 7). However, AC3-I x PLN-/- mice had a trend (P = 0.07) toward greater apoptosis with Iso than did AC3-I mice with PLN expression (compare Fig. 7B and Fig. 1B), suggesting that PLN contributes to the antiapoptotic mechanism of CaMKII inhibition. Control experiments with saline showed no difference (P = 0.6) in myocyte apoptosis in AC3-I x PLN–/– and AC3-C x PLN–/– hearts (Fig. 7). These findings show that CaMKII inhibition can reduce apoptosis during beta-AR stimulation even in the absence of PLN, but they leave open the possibility that PLN participates in the antiapoptotic effects of myocardial CaMKII inhibition.


Figure 7
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  		Fig. 7. PLN-independent protection from Iso-mediated apoptosis by CaMKII inhibition. A: photomicrographs show reduced TUNEL-positive staining in AC3-I x PLN–/– compared with AC3-C x PLN–/– LV transverse sections 2 h after Iso injection. Calibration bar indicates 15 µm. B: summary data for TUNEL-positive staining in Iso-treated hearts (6–7 hearts/group) and saline-treated hearts (3–4 hearts/group). Iso significantly ({dagger}P < 0.001) increased TUNEL staining in AC3-C x PLN–/– hearts compared with saline, whereas the AC3-I x PLN–/– hearts had less TUNEL staining than did the AC3-C x PLN–/– hearts (*P = 0.05).

 
In contrast to the persistent protective effects of CaMKII inhibition on Iso-mediated apoptosis in the absence of PLN, the protective effect of CaMKII inhibition on apoptosis after MI was eliminated in PLN–/– hearts (Fig. 8, A and B). There was no difference in the surgically induced ischemic areas between AC3-I x PLN–/– and AC3-C x PLN–/– mice (Fig. 8B). These findings suggest that PLN is important for the antiapoptotic actions of CaMKII inhibition during ischemia.


Figure 8
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  		Fig. 8. CaMKII inhibition fails to protect against apoptosis after MI in the absence of PLN. A: photomicrographs (arranged as in Figs. 1, 2, 3, 5, and 7) show similar amounts of TUNEL-positive staining in AC3-I x PLN–/– compared with AC3-C x PLN–/– LV transverse sections 2 h after MI. Calibration bar indicates 15 µm. B: summary data show equivalent TUNEL staining in AC3-I x PLN–/– hearts compared with AC3-C x PLN–/– hearts (3–4 hearts/group) 2 h after MI. C: ischemic areas were similar in AC3-I x PLN–/– and AC3-C x PLN–/– hearts, as assessed by percent area of TUNEL staining.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results add to a growing body of evidence that CaMKII can be recruited as a pathological signal in heart (1, 41). Catecholamine-stimulated Ca2+ overload is a cause of cardiomyocyte death, but diverse signaling pathways potentially respond to changes in cellular Ca2+ (37). Recently, in vitro studies showed that CaMKII can play a critical role in SR Ca2+ overload-related myocyte apoptosis induced by Iso treatment (43). Our study extends these earlier findings in cultured cardiomyocytes by showing that CaMKII inhibition reduces ischemia-induced and prevents catecholamine-induced myocyte apoptosis in vivo, indicating that CaMKII is an important pathophysiological mediator of apoptosis. Our findings suggest that improved cardiac function originally reported in AC3-I mice after excessive Iso stimulation and MI (40) is, in part, due to reduced apoptosis. These results further suggest that CaMKII inhibition may be an important therapeutic approach for reducing myocyte loss in ischemia and structural heart disease. Cultured cell systems offer significant experimental advantages but exist under starkly different conditions than do working myocytes in situ. Our findings provide a new rationale for utilizing both in vitro and in vivo approaches in studies of CaMKII signaling, because CaMKII inhibition was successful in preventing or reducing apoptosis in response to Iso in vivo as well as in cultured adult ventricular myocytes (43).

MI is a major cause of structural heart disease and mortality worldwide (18). Currently, the most effective treatments to reduce cell death and adverse remodeling from MI involve acute restoration of coronary blood flow within 6 h of occlusion (6). Thus interventions that effectively delay or reduce myocyte loss early in MI have the potential to improve the efficacy of reperfusion therapies by lengthening the time window for successful reperfusion and increasing the number of surviving cardiomyocytes. Our finding that CaMKII inhibition significantly delays myocyte apoptosis during MI, in a time frame that is important in patients, suggests that CaMKII inhibition could improve currently available therapies for myocardial ischemia. It will be important to repeat these experiments in larger animals with heart rates that are more similar to that of humans to understand whether the present findings are truly relevant to patients. A lack of highly specific CaMKII inhibitory drugs was one difficulty that motivated us to make transgenic mice with myocardial CaMKII inhibition (1). However, it will be necessary to develop CaMKII inhibitory drugs with improved specificity to perform studies in larger animals where genetic approaches to modulating CaMKII are less tractable than in mice.

Overload of intracellular Ca2+ endoplasmic reticulum stores is an important stimulus for apoptosis in nonmuscle cells (9), but the potential relationship between SR Ca2+ overload in cardiomyocytes and apoptosis is incompletely understood, in part because previous work has not evaluated these cellular components under physiological in vivo conditions. Our finding that PLN ablation enhances susceptibility to apoptosis and verapamil eliminates this susceptibility indicates that SR Ca2+ overload contributes to beta-AR-mediated apoptosis in the working heart. CaMKII inhibition significantly protects against SR Ca2+ overload (40), whereas PLN ablation eliminates differences in SR Ca2+ content between AC3-I- and AC3-C-expressing hearts (35). PLN ablation appears to reduce the beneficial effects of CaMKII inhibition on apoptosis during beta-AR stimulation and prevents protection of CaMKII inhibition against apoptosis during MI.

A potentially important implication of our findings is that efforts to target repletion of SR calcium content to improve mechanical function during heart failure may increase apoptosis. One characteristic change in failing human and animal myocytes is reduced SR calcium content that can contribute to depressed myocyte contractility (26, 27). Reduced SR Ca2+ is thus a potential target for therapy by inotropic drugs, by PLN knockout, or by SERCA2a overexpression. Although some studies have shown promise (14, 23, 29), others have shown worsened cardiomyopathy (16, 30, 32) by PLN ablation. A human mutation producing PLN that is incapable of negatively regulating SERCA2a results in severe cardiomyopathy and heart failure (16), suggesting that SR Ca2+ repletion could have negative consequences in patients. Our findings that PLN ablation can increase apoptosis during Iso provide a potential mechanism for understanding adverse outcomes related to therapeutic repletion of SR Ca2+ content.

The mechanisms of Ca2+ enhancement in MI and during isoproterenol are likely to be different. For example, the Na+/Ca2+ exchanger is implicated in Ca2+ overload during MI (22), whereas increased L-type Ca2+ current may be preeminent during catecholamine stimulation (39). Thus it will be important for future studies to consider how differences in these and other cellular Ca2+ mobilizing mechanisms may affect CaMKII signaling. CaMKII inhibition reduces apoptosis 2 h (Fig. 2) and 5 h (Fig. 3) after MI and preserves LV function from 1 to 3 wk after MI (40). These findings support the idea that CaMKII inhibition could be a novel and effective therapy during ischemia. On the other hand, CaMKII activity may be beneficial for acute recovery of contractile function in ischemia-reperfusion by PLN phosphorylation (24). Furthermore, CaMKII activity may have physiological importance for controlling dynamic mechanical responses in heart (11). At present, it is unknown whether the early recovery of cellular mechanical function is beneficial or detrimental to cell survival.

Myocardial responses to CaMKII are highly dependent on the duration of increased CaMKII activity or CaMKII inhibition. CaMKII inhibition reduces L-type Ca2+ channel facilitation within minutes (2, 36, 38), and the addition of activated CaMKII to excised cardiomyocyte membranes induces L-type Ca2+ channels to enter a highly active gating mode within seconds (13). However, CaMKII inhibition in AC3-I mice does not impair basal contractile function or acute responses to Iso in isolated ventricular myocytes compared with AC3-C and WT cells (40), suggesting that chronic CaMKII inhibition (i.e., from the time of birth) does not significantly affect acute "fight or flight" responses to catecholamine stimulation in the heart. In contrast, CaMKII activation is required for maintaining normal cardiomyocyte contraction under conditions of prolonged (24 h) catecholamine exposure (34), suggesting that CaMKII can be critical for physiological stress responses in the heart. This concept is supported by findings that CaMKII overexpression (24–48 h) increases L-type Ca2+ current and enhances Ca2+ release from the SR (20). Truly chronic myocardial CaMKII overexpression, genetically induced from the time of birth, recapitulates important aspects of structural heart disease, including cardiomyocyte hypertrophy, premature death, and reduced contractile function (21, 42). Our findings that CaMKII is important for signaling apoptosis within 2 h after Iso and 2–5 h after MI suggest that "early" CaMKII activation may be important for initiating adverse LV remodeling responses that lead to heart failure and sudden death.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
We acknowledge support from National Heart, Lung, and Blood Institute (HL-070250, HL-62494, and HL-046681).


   ACKNOWLEDGMENTS
 
M. E. Anderson is an Established Investigator of the American Heart Association. We are grateful for helpful discussions with Dr. Elizabeth Yang.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. E Anderson, Dept. of Medicine, Carver College of Medicine, Univ. of Iowa, Iowa City, IA 52242 (e-mail: mark-e-anderson{at}uiowa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anderson ME. Calmodulin kinase signaling in heart: an intriguing candidate target for therapy of myocardial dysfunction and arrhythmias. Pharmacol Ther 106: 39–55, 2005.[CrossRef][ISI][Medline]
  2. Anderson ME, Braun AP, Schulman H, and Premack BA. Multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-induced enhancement of the L-type Ca2+ current in rabbit ventricular myocytes. Circ Res 75: 854–861, 1994.[Abstract/Free Full Text]
  3. Benedict CR, Shelton B, Johnstone DE, Francis G, Greenberg B, Konstam M, Probstfield JL, and Yusuf S. Prognostic significance of plasma norepinephrine in patients with asymptomatic left ventricular dysfunction. SOLVD Investigators. Circulation 94: 690–697, 1996.[Abstract/Free Full Text]
  4. Berridge MJ, Bootman MD, and Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529, 2003.[CrossRef][ISI][Medline]
  5. Bilezikjian LM, Kranias EG, Potter JD, and Schwartz A. Studies on phosphorylation of canine cardiac sarcoplasmic reticulum by calmodulin-dependent protein kinase. Circ Res 49: 1356–1362, 1981.[Abstract/Free Full Text]
  6. Bonnefoy E, Lapostolle F, Leizorovicz A, Steg G, McFadden EP, Dubien PY, Cattan S, Boullenger E, Machecourt J, Lacroute JM, Cassagnes J, Dissait F, and Touboul P. Primary angioplasty versus prehospital fibrinolysis in acute myocardial infarction: a randomised study. Lancet 360: 825–829, 2002.[CrossRef][ISI][Medline]
  7. Braun AP and Schulman H. The multifunctional calcium/calmodulin-dependent protein kinase: from form to function. Annu Rev Physiol 57: 417–445, 1995.[CrossRef][ISI][Medline]
  8. Chesley A, Lundberg MS, Asai T, Xiao RP, Ohtani S, Lakatta EG, and Crow MT. The beta2-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through Gi-dependent coupling to phosphatidylinositol 3'-kinase. Circ Res 87: 1172–1179, 2000.[Abstract/Free Full Text]
  9. Danial NN and Korsmeyer SJ. Cell death: critical control points. Cell 116: 205–219, 2004.[CrossRef][ISI][Medline]
  10. De Koninck P and Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science 279: 227–230, 1998.[Abstract/Free Full Text]
  11. DeSantiago J, Maier LS, and Bers DM. Frequency-dependent acceleration of relaxation in the heart depends on CaMKII, but not phospholamban. J Mol Cell Cardiol 34: 975–984, 2002.[CrossRef][ISI][Medline]
  12. Dumont EA, Hofstra L, van Heerde WL, van den Eijnde S, Doevendans PA, DeMuinck E, Daemen MA, Smits JF, Frederik P, Wellens HJ, Daemen MJ, and Reutelingsperger CP. Cardiomyocyte death induced by myocardial ischemia and reperfusion: measurement with recombinant human annexin-V in a mouse model. Circulation 102: 1564–1568, 2000.[Abstract/Free Full Text]
  13. Dzhura I, Wu Y, Colbran RJ, Balser JR, and Anderson ME. Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels. Nat Cell Biol 2: 173–177, 2000.[CrossRef][ISI][Medline]
  14. Engelhardt S, Hein L, Dyachenkow V, Kranias EG, Isenberg G, and Lohse MJ. Altered calcium handling is critically involved in the cardiotoxic effects of chronic beta-adrenergic stimulation. Circulation 109: 1154–1160, 2004.[Abstract/Free Full Text]
  15. European Myocardial Infarction Project Group. Prehospital thrombolytic therapy in patients with suspected acute myocardial infarction. N Engl J Med 329: 383–389, 1993.[Abstract/Free Full Text]
  16. Haghighi K, Kolokathis F, Pater L, Lynch RA, Asahi M, Gramolini AO, Fan GC, Tsiapras D, Hahn HS, Adamopoulos S, Liggett SB, Dorn GW, MacLennan DH, Kremastinos DT, and Kranias EG. Human phospholamban null results in lethal dilated cardiomyopathy revealing a critical difference between mouse and human. J Clin Invest 111: 869–876, 2003.[CrossRef][ISI][Medline]
  17. Hoch B, Meyer R, Hetzer R, Krause EG, and Karczewski P. Identification and expression of delta-isoforms of the multifunctional Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human myocardium. Circ Res 84: 713–721, 1999.[Abstract/Free Full Text]
  18. Jessup M and Brozena S. Heart failure. N Engl J Med 348: 2007–2018, 2003.[Free Full Text]
  19. Kiss E, Edes I, Sato Y, Luo W, Liggett SB, and Kranias EG. beta-Adrenergic regulation of cAMP and protein phosphorylation in phospholamban-knockout mouse hearts. Am J Physiol Heart Circ Physiol 272: H785–H790, 1997.[Abstract/Free Full Text]
  20. Kohlhaas M, Zhang T, Seidler T, Zibrova D, Dybkova N, Steen A, Wagner S, Chen L, Brown JH, Bers DM, and Maier LS. Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes. Circ Res 98: 235–244, 2006.[Abstract/Free Full Text]
  21. Maier LS, Zhang T, Chen L, DeSantiago J, Brown JH, and Bers DM. Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release. Circ Res 92: 904–911, 2003.[Abstract/Free Full Text]
  22. Mattiazzi A, Mundina-Weilenmann C, Vittone L, and Said M. Phosphorylation of phospholamban in ischemia-reperfusion injury: functional role of Thr17 residue. Mol Cell Biochem 263: 131–136, 2004.[CrossRef][ISI][Medline]
  23. Minamisawa S, Hoshijima M, Chu G, Ward CA, Frank K, Gu Y, Martone ME, Wang Y, Ross JJ, Kranias EG, Giles WR, and Chien KR. Chronic phospholamban-sarcoplasmic reticulum calcium ATPase interaction is the critical calcium cycling defect in dilated cardiomyopathy. Cell 99: 313–322, 1999.[CrossRef][ISI][Medline]
  24. Mundina-Weilenmann C, Ferrero P, Said M, Vittone L, Kranias EG, and Mattiazzi A. Role of phosphorylation of Thr17 residue of phospholamban in mechanical recovery during hypercapnic acidosis. Cardiovasc Res 66: 114–122, 2005.[CrossRef][ISI][Medline]
  25. Olivetti G, Quaini F, Sala R, Lagrasta C, Corradi D, Bonacina E, Gambert SR, Cigola E, and Anversa P. Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol 28: 2005–2016, 1996.[CrossRef][ISI][Medline]
  26. Piacentino V III, Weber CR, Chen X, Weisser-Thomas J, Margulies KB, Bers DM, and Houser SR. Cellular basis of abnormal calcium transients of failing human ventricular myocytes. Circ Res 92: 651–658, 2003.[Abstract/Free Full Text]
  27. Pieske B, Maier LS, Bers DM, and Hasenfuss G. Ca2+ handling and sarcoplasmic reticulum Ca2+ content in isolated failing and nonfailing human myocardium. Circ Res 85: 38–46, 1999.[Abstract/Free Full Text]
  28. Rottman JN, Ni G, Khoo M, Wang Z, Zhang W, Anderson ME, and Madu EC. Temporal changes in ventricular function assessed echocardiographically in conscious and anesthetized mice. J Am Soc Echocardiogr 16: 1150–1157, 2003.[CrossRef][ISI][Medline]
  29. Sato Y, Kiriazis H, Yatani A, Schmidt AG, Hahn H, Ferguson DG, Sako H, Mitarai S, Honda R, Mesnard-Rouiller L, Frank KF, Beyermann B, Wu G, Fujimori K, Dorn GW, and Kranias EG. Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation. J Biol Chem 276: 9392–9399, 2001.[Abstract/Free Full Text]
  30. Schmitt JP, Kamisago M, Asahi M, Li GH, Ahmad F, Mende U, Kranias EG, MacLennan DH, Seidman JG, and Seidman CE. Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science 299: 1410–1413, 2003.[Abstract/Free Full Text]
  31. Schomig A. Catecholamines in myocardial ischemia. Systemic and cardiac release. Circulation 82: II13–II22, 1990.
  32. Song Q, Schmidt AG, Hahn HS, Carr AN, Frank B, Pater L, Gerst M, Young K, Hoit BD, McConnell BK, Haghighi K, Seidman CE, Seidman JG, Dorn GW, and Kranias EG. Rescue of cardiomyocyte dysfunction by phospholamban ablation does not prevent ventricular failure in genetic hypertrophy. J Clin Invest 111: 859–867, 2003.[CrossRef][ISI][Medline]
  33. Vinogradova TM, Zhou YY, Bogdanov KY, Yang DM, Kuschel M, Cheng HP, and Xiao RP. Sinoatrial node pacemaker activity requires Ca2+/calmodulin-dependent protein kinase II activation. Circ Res 87: 760–767, 2000.[Abstract/Free Full Text]
  34. Wang W, Zhu W, Wang S, Yang D, Crow MT, Xiao RP, and Cheng H. Sustained beta1-adrenergic stimulation modulates cardiac contractility by Ca2+/calmodulin kinase signaling pathway. Circ Res 95: 798–806, 2004.[Abstract/Free Full Text]
  35. Wu Y, Shintani A, Grueter C, Zhang R, Hou Y, Yang J, Kranias EG, Colbran RJ, and Anderson ME. Suppression of dynamic Ca2+ transient responses to pacing in ventricular myocytes from mice with genetic calmodulin kinase II inhibition. J Mol Cell Cardiol 40: 213–23, 2006.[CrossRef][ISI][Medline]
  36. Xiao RP, Cheng H, Lederer WJ, Suzuki T, and Lakatta EG. Dual regulation of Ca2+/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. Proc Natl Acad Sci USA 91: 9659–9663, 1994.[Abstract/Free Full Text]
  37. Yano M, Ikeda Y, and Matsuzaki M. Altered intracellular Ca2+ handling in heart failure. J Clin Invest 115: 556–564, 2005.[CrossRef][ISI][Medline]
  38. Yuan W and Bers DM. Ca-dependent facilitation of cardiac Ca current is due to Ca-calmodulin-dependent protein kinase. Am J Physiol Heart Circ Physiol 267: H982–H993, 1994.[Abstract/Free Full Text]
  39. Yue DT, Herzig S, and Marban E. Beta-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc Natl Acad Sci USA 87: 753–757, 1990.[Abstract/Free Full Text]
  40. Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, and Anderson ME. Calmodulin kinase II inhibition protects against structural heart disease. Nat Med 11: 409–417, 2005.[CrossRef][ISI][Medline]
  41. Zhang T and Brown JH. Role of Ca2+/calmodulin-dependent protein kinase II in cardiac hypertrophy and heart failure. Cardiovasc Res 63: 476–486, 2004.[Abstract/Free Full Text]
  42. Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J Jr, Bers DM, and Brown JH. The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ Res 92: 912–919, 2003.[Abstract/Free Full Text]
  43. Zhu WZ, Wang SQ, Chakir K, Yang DM, Zhang T, Brown JH, Devic E, Kobilka BK, Cheng HP, and Xiao RP. Linkage of beta1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II. J Clin Invest 111: 617–625, 2003.[CrossRef][ISI][Medline]



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